Antibiotic 31F508 ALPHA1, ALPHA2, BETA1 and BETA2

ABSTRACT

This invention relates to antibiotic 31F508α 1  of the formula ##STR1## 31F508α 2 , 31F508β 1  and 31F508β 2 , derived from the microorganism Streptomyces viridodiastaticus subsp. &#34;littus&#34; which are useful as anti-bacterial agents.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to new antibiotics designated 31F508α₁, 31F508α₂, 31F508β₁ and 31F508β₂, to their production by fermentation, to methods for their recovery and concentration from crude solutions and to processes for their purification. The present invention includes within its scope the agents in dilute form, as crude concentrates, as a complex of all components, in pure form as individual components and a novel strain of Streptomyces viridodiastaticus subsp. "littus".

2. Description of the Prior Art

Naphthyridinomycin antibiotics are reported in U.S. Pat. No. 4,003,902.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. I shows a proton magnetic resonance spectrum of 31F508α₁.

FIG. II shows an ultraviolet absorption spectrum of 31F508α₂.

FIG. III shows an infrared absorption spectrum of 31F508α₂.

FIG. IV shows a proton magnetic resonance spectrum of 31F508α₂.

FIG. V shows a carbon-13 nuclear magnetic resonance spectrum of 31F508α₂.

FIG. VI shows a proton magnetic resonance spectrum of 31F508β₂

FIG. VII shows a proton magnetic resonance spectrum of 31F508β₂

FIG. VIII shows a carbon-13 nuclear magnetic resonance spectrum

DESCRIPTION OF THE PREFERRED EMBODIMENTS

New antibiotics designated 31F508α₁, 31F508α₂, 31F508β₁ and 31F508β₂ have now been found. The structure of new antibiotic 31F508α₁ is: ##STR2## The physico-chemical characteristics of 31F508α₁ are as follows:

31F508α₁

1. Molecular weight: 387 (FABMS);

2. Apparent molecular formula:

C₂₀ H₂₅ N₃ O₅

3. Proton nuclear magnetic resonance spectrum: as shown in FIG. I.

The structure of the new antibiotic 31F508α₂ is: ##STR3## The physico-chemical characteristics of 31F508α₂ are as follows:

31F508α₂

1. Molecular weight: 401(FABMS);

2. Apparent molecular formula:

C₂₁ H₂₇ N₃ O₅

3. Ultraviolet absorption spectrum: as shown in FIG. II 292NM (n2729) in methyl alcohol.

4. Infrared absorption spectrum as shown in FIG. III (cm⁻¹) (KBr disc)

3353, 3026, 2967, 2626, 1662, 1623, 1467, 1429, 1371, 1334, 1270, 1189, 1154, 1120, 1061, 1004, 965, 933.

5. Proton nuclear magnetic resonance spectrum: as shown in FIG. IV (300 MHz, D₂ O/TFA) ppm

2.08, 2.19(3H, s), 2.71, 2.95(3H, s), 3.01, 3.47, 3.62, 3.69, 3.76(3H, s), 3.831, 3.84, 3.98, 4.1, 4.28, 4.29, 4.43, 4.439, 4.66(1H, s), 4.83(1H, s), 5.08, 5.43 (1H, s), 5.52.

6. Carbon-13 nuclear magnetic resonance spectrum: as shown in FIG. V (D₂ O/TFA) ppm

10.06, 26.37, 32.3, 40.02, 47.67, 50.67, 52.47, 54.46, 60.73, 61.54, 61.6, 64.48, 68.11, 78.15, 92.85, 115.03, 120.51, 121.04, 141.09, 145.76, 148.77, 177.49.

The structure of the new antibiotic 31F508β₁ is: ##STR4## The physico-chemical characteristics of 31F508β₁ are as follows: 31F508β₁

1. Molecular weight: 385(FABMS);

2. Apparent molecular formula:

C₂₀ H₂₃ N₃ O₅

The structure of the new antibiotic 31F508b₂ is ##STR5## The physico-chemical characteristics of 31F508β₂ are as follows: 31F508β₂

1. Molecular weight: 399(FABMS);

2. Apparent molecular formula:

C₂₁ H₂₅ N₃ O₅

3. Ultraviolet absorption spectrum: as shown in FIG. VI. 270NM (n=10508);

4. Proton nuclear magnetic resonance spectrum as shown in FIG. VII.

1.63, 1.94(3H5), 2.3, 2.35(3H5), 2.81,2.95, 3.01, 3.05, 3.15, 3.56, 3.7, 3.72, 3.93, 4.0(3H5), 4.02, 4.42, 4.73(1H5)

5. Carbon-13 nuclear magnetic resonance spectrum as shown in FIG. VIII.

8.77, 28.69, 33.96, 41.09, 47.87, 50.16, 52.08, 54.24, 60.28, 60.87, 61.71, 65.8, 78.77, 93.62 128.0, 142.59, 142.9, 155.85, 181.48, 186.76

The new antibacterial agents 31F508α₁, 31F508α₂, 31F508β₁ and 31F508β₂ are formed during the cultivation under controlled conditions of a new strain of Streptomyces viridodiastaticus subsp. "littus".

This microorganism is maintained in the culture collection of the Medical Research Division, American Cyanamid Company, Pearl River, N.Y. 10965 as culture 31F508. A viable culture of this new microorganism has been deposited with the Patent Culture Collection Laboratory, Northern Regional Research Center, U.S. Department of Agriculture, Peoria, Ill. 61604, and has been added to its permanent collection. It has been assigned the strain designation NRRL-21082 by said depository. Access to said culture, under strain designation NRRL-21082, during pendency of the instant application, shall be available to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R.=1.14 and 35 U.S.C.=122, and all restrictions on availability to the public of such culture will be irrevocably removed upon grant of a patent on the instant application.

Culture 31F508 is isolated from a shoreline soil sample taken from Key West, Fla.

Culture 31F508 has the following cultural characteristics as described and compared to Streptomyces viridodiasticus for taxonomic identification in Table I.

The macromorphology for culture 31F508 is described and compared to Streptomyces viridodiasticus in Table II.

                  TABLE I                                                          ______________________________________                                         CULTURAL CHARACTERISTICS                                                       Characteristic  31F508     S. viridodiastaticus                                ______________________________________                                         Aerial Mycelium Rectiflexibiles                                                                           Rectiflexibiles                                     Fragmentation of                                                                               None       None                                                Substrate Mycelium                                                             Zoospores and Sporangia                                                                        None       None                                                Spore Chain     15-20      15-20                                               Spore Shape     Oval       Oval                                                Spore Surface   Spiny      Spiny                                               DAP Analysis    LL-DAP     LL-DAP                                              ______________________________________                                    

                  TABLE II                                                         ______________________________________                                         MACROPHORPHOLOGY                                                               Medium   31F508 Morphology.sup.a                                                                           S. viridodiastaticus                               ______________________________________                                         Yeast-Malt                                                                              G:     Abundant       Abundant                                        (ISP2)   AM:    Medium Gray (256)                                                                             Medium Gray (256)                                        SM:    Gray Yellow-Brown                                                                             Medium                                                          (80,81)        Olive-Brown (95)                                         SP:    None           None                                            Oatmeal  G:     Abundant       Abundant                                        (ISP3)   AM:    Medium Gray (265)                                                                             Medium Gray (265)                                        SM:    Light Olive-Gray                                                                              Dark Gray to Dark                                               (112)          Yellow (266,88)                                          SP:    None           None                                            Inorganic                                                                               G:     Abundant       Abundant                                        Salts - Starch                                                                          AM:    Light to Medium                                                                               Medium Gray (265)                               (ISP4)          Gray (264,265)                                                          SM:    Medium Gray to Medium Gray (265)                                               Gray Yellow (265,90)                                                    SP:    None           None                                            Glycerol -                                                                              G:     Moderate       Moderate                                        Asparagine                                                                              AM:    Medium Gray (265)                                                                             Medium Gray (265)                               (ISP5)   SM:    Colorless      Colorless                                                SP:    None           None                                            ______________________________________                                          G, growth; AM, aerial mycelium; SM, subtrate mycelium; SP, soluble pigmen      .sup.a ISCC, National Bureau of Standard Centroid Color Charts,                Publication 440, Washington, D.C., 1976.                                 

The physiological reactions of 31F508 are shown and compared to Streptomyces viridodiasticus in Table III.

                  TABLE III                                                        ______________________________________                                         PHYSIOLOGICAL REACTIONS                                                                        31F508 S. viridodiastaticus                                    ______________________________________                                         Utilization of Carbon Sources:                                                 D-Glucose         +        +                                                   L-Arabinose       +        +                                                   Sucrose           +        +                                                   D-Xylose          +        +                                                   Inositol          +        +                                                   D-Mannitol        +        +                                                   β-D-Fructose +        +                                                   α-L-Rhamnose                                                                               +        +                                                   Raffinose         -        -                                                   Cellulose         -        -                                                   Hydrolysis of:                                                                 Casein            +        +                                                   Xanthine          +        +                                                   Hypoxanthine      +        +                                                   Tyrosine          +        +                                                   Adenine           -        -                                                   Esculin           -        -                                                   Gelatin           +        +                                                   Starch            +        +                                                   Production of:                                                                 Urease            +        -                                                   Melanin           -        -                                                   Phosphatase       +        +                                                   Decarboxylation of:                                                            Acetate           +        +                                                   Benzoate          -        -                                                   Citrate           +        +                                                   Lactate           +        ±                                                Malate            +        +                                                   Mucate            -        -                                                   Oxalate           +        -                                                   Proprionate       +        +                                                   Pyruvate          +        +                                                   Succinate         +        +                                                   Tartrate          -        -                                                   Acid Production from:                                                          Arabinose         -        +                                                   Dulcitol          -        -                                                   Erythritol        -        -                                                   Glucose           +        +                                                   Inositl           +        +                                                   Lactose           +        +                                                   Mannitol          +        +                                                   Mannose           +        +                                                   Methyl-α -D-glucoside                                                                      +        +                                                   Melibiose         +        +                                                   Raffinose         ±     -                                                   α-L-Rhamnose                                                                               +        +                                                   Sorbitol          -        -                                                   Trehalose         +        +                                                   Resistance to                                                                  Lysozyme          -        -                                                   Growth at                                                                      22° C.     +        +                                                   28° C.     +        +                                                   45° C.     -        +                                                   50° C.     -        +                                                   Growth on NaCl                                                                 5%                +        +                                                   6%                +        ±                                                8%                +        ±                                                ______________________________________                                          +: positive, -: negative, ±: weak                                     

It is to be understood that for production of these new antibacterial agents the present invention is not limited to this particular organism or to organisms fully answering the above characteristics which are given for illustrative purposes only. In fact, it is desired and intended to include the use of mutants produced from this organism by various means such as exposure to X-radiation, ultraviolet radiation, N'-methyl-N'-nitro-N-nitrosoguanidine, actinophages and the like.

The in vitro antibacterial activity of 31F508α₁ and α2 is determined against a spectrum of gram-positive and gram-negative bacteria by a standard agar dilution method. Mueller-Hinton agar with and without 5% sheep blood and two-fold decreasing concentrations of either 31F508α₁ or α₂ are poured into petri dishes. The agar surfaces are inoculated with 1 to 5×10⁴ colony forming units of bacteria by means of the Steers replicating units of bacteria by means of the Steers replicating device. The lowest concentration of antibiotic that inhibits growth of a bacterial strain after 18 hours incubation is recorded as the minimal inhibitory concentration for that strain. The results are given in Table IV. The results are given in Table V with sheeps blood added to the agar.

                  TABLE IV                                                         ______________________________________                                         In vitro Antibacterial Activity of                                             31F508α.sub.1 and 31F508α.sub.2                                                      Minimum Inhibitory                                                             Concentration mcg/ml                                         Organism            31F508α.sub.1                                                                      31F508α.sub.2                              ______________________________________                                          1.  Staphylococcus aureus                                                                             0.030     0.004                                             (MEMC-89-4)                                                                2.  Staphylococcus aureus                                                                             0.030     0.004                                             (ID-2371)                                                                  3.  Staphylococcus aureus                                                                             0.008     0.002                                             (ID-2727)                                                                  4.  Staphylococcus aureus                                                                             0.004     0.002                                             (SMITH)                                                                    5.  Staphylococcus aureus                                                                             0.030     0.004                                             (ID-3105)                                                                  6.  Staphylococcus aureus                                                                             0.030     0.004                                             (ID-4379)                                                                  7.  Staphylococcus aureus                                                                             0.060     0.008                                             (ATCC 29213)                                                               8.  Staphylococcus haemolyticus                                                                       0.008     0.002                                             (ID-4061)                                                                  9.  CNS (ID-3135)      0.015     0.002                                        10.  CNS (ID-3276)      0.008     0.002                                        11.  CNS (ID-3120)      0.015     0.002                                        12.  CNS (ID-3941)      0.030     0.002                                        13.  CNS (4615)         0.015     0.002                                        14.  E. faecalis (ID-4168)                                                                             0.060     0.120                                        15.  E. faecalis (ID-1829)                                                                             0.120     0.060                                        16.  E. faecalis (ID-2131)                                                                             0.120     0.250                                        17.  E. faecalis (12201)                                                                               0.030     0.015                                        18.  E. faecalis (ATCC 29212)                                                                          0.060     0.060                                        19.  E. faecium (12202) 0.030     0.015                                        20.  E. faecium (ID-3301)                                                                              0.060     0.120                                        21.  E. faecium  (ID-4133)                                                                             0.002     0.002                                        22.  E. Avium (ID-3953) 0.060     0.030                                        23.  S. Pyogenes (ID-3187)                                                                             0.002     0.002                                        24.  S. agalactiae (ID-4079)                                                                           0.004     0.004                                        25.  Streptococcus pneumoniae                                                                          0.002     0.002                                             (ID-4444)                                                                 26.  P. aeruginosa (ATCC 27853)                                                                        2.000     2.000                                        27.  M. morganii (VGH 84-11)                                                                           1.000     0.500                                        28.  E. coli (J2175)    1.000     1.000                                        29.  E. coli (J2445)    0.060     0.004                                        30.  E. coli (ATCC 25922)                                                                              1.000     0.500                                        31.  B. cereus (Bacto)  0.500     0.250                                        32.  S. lutea (ATCC 9341)                                                                              0.002     0.002                                        ______________________________________                                    

                  TABLE V                                                          ______________________________________                                         In vitro Antibacterial Activity of                                             31F508α.sub.1 and 31F508α.sub.2                                    In the Presence of Sheeps Blood                                                                  Minimum Inhibitory                                                             Concentration mcg/ml                                         Organism            31F508α.sub.1                                                                      31F508β.sub.2                               ______________________________________                                         1.   Staphylococcus aureus                                                                             0.060    0.060                                              (MEMC-89-4)                                                                2.  Staphylococcus aureus                                                                             0.060    0.030                                              (ID-2371)                                                                  3.  Staphylococcus aureus                                                                             0.015    0.015                                              (ID-2727)                                                                  4.  Staphylococcus aureus                                                                             0.008    0.002                                              (SMITH)                                                                    5.  Staphylococcus aureus                                                                             0.060    0.060                                              (ID-3105)                                                                  6.  Staphylococcus aureus                                                                             0.120    0.060                                              (ID-4379)                                                                  7.  Staphylococcus aureus                                                                             0.120    0.060                                              (ATCC 29213)                                                               8.  Staphylococcus haemolyticus                                                                       0.015    0.004                                              (ID-4061)                                                                  9.  CNS (ID-3135)      0.030    0.015                                         10.  CNS (ID-3276)      0.030    0.015                                         11.  CNS (ID-3120)      0.030    0.030                                         12.  CNS (ID-3941)      0.060    0.015                                         13.  CNS (4615)         0.030    0.015                                         14.  E. faecalis (ID-4168)                                                                             0.120    0.500                                         15.  E. faecalis (ID-1829)                                                                             0.500    0.500                                         16.  E. faecalis (ID-2131)                                                                             0.250    0.500                                         17.  E. faecalis (12201)                                                                               0.06     0.120                                         18.  E. faecalis (ATCC 29212)                                                                          0.120    0.500                                         19.  E. faecium (12202) 0.250    0.250                                         20.  E. faecium (ID-3301)                                                                              0.120    0.500                                         21.  E. faecium (ID-4133)                                                                              0.002    0.002                                         22.  E. Avium (ID-3953) 0.250    0.500                                         23.  S. Pyogenes (ID-3187)                                                                             0.004    0.030                                         24.  S. agalactiae (ID-4079)                                                                           0.015    0.015                                         25.  Streptococcus pneumoniae                                                                          0.008    0.002                                              (ID-4444)                                                                 26.  P. aeruginosa (ATCC 27853)                                                                        2.000    4.000                                         27.  M. morganii (VGH 84-11)                                                                           2.000    2.000                                         28.  E. coli (J2175)    2.000    2.000                                         29.  E. coli (J2445)    0.120    0.030                                         30.  E. coli (ATCC 25922)                                                                              1.000    1.000                                         31.  B. cereus (Bacto)  1.000    1.000                                         32.  S. lutea (ATCC 9341)                                                                              0.002    0.002                                         ______________________________________                                    

The in vivo antibacterial activity of antibiotic 31F508α₂ is established by infecting female CD-1 mice from Charles River Laboratories, weighing 20+2 g each, intraperitoneally of sufficient bacterial cells suspended in either broth or 5% hog gastric mucin to kill non-treated controls within 24-48 hours. Two-fold serially diluted doses of the antibacterial agent contained in phosphate buffered saline (PBS, pH 7.4, 0.01M) are administered intravenously (0.2 ml) or contained in 0.2% aqueous agar, subcutaneously (0.5 ml) or orally (0.5 ml) one half hour after infection. In each test, five mice are treated per dose level. The results of this test appear in Table VI.

                  TABLE VI                                                         ______________________________________                                         In vivo Activity of 31F508α.sub.2                                        Against Escherichia coli #311                                                  Route     Dose Levels (Mg/Kg)                                                                           Survival Ratios                                       ______________________________________                                         SSC       8              0/5                                                             4              0/5                                                             2              0/5                                                             1              0/5                                                             0.5            0/5                                                             0.25           0/5                                                   SOD       8              0/5                                                             4              0/5                                                             2              0/5                                                             1              0/5                                                             0.5            0/5                                                             0.25           0/5                                                   SIV       4              0/5                                                             2              0/5                                                             1              0/5                                                             0.5            0/5                                                             0.25           0/5                                                   ______________________________________                                    

In therapeutic use, the compounds of this invention may be administered in the form of conventional pharmaceutical compositions appropriate for the intended use. Such compositions may be formulated so as to be suitable for oral, parenteral or topical administration. The active ingredient may be combined in admixture with a non-toxic pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration, i.e., oral, parenteral or topical.

GENERAL FERMENTATION CONDITIONS

Cultivation of Streptomyces viridodiastaticus subsp. "littus". 31F508 may be carried out in a wide variety of liquid culture media. Media which are useful for the production of 31F508 include an assimilable source of carbon, such as dextrin, sucrose, molasses, glycerol, etc.; an assimilable source of nitrogen, such as protein, protein hydrolysate, polypeptides, amino acids, corn steep liquor, etc.; and inorganic anions and cations, such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc., are supplied as impurities of other constituents of the media. Aeration in tanks and bottles is supplied by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanical impeller. An antifoam agent such as silicon oil may be added as needed.

GENERAL PROCEDURE FOR THE ISOLATION OF 31F508α₁, 31F508α₂, 31F508β₁ AND 31F508β₂

The 31F508α₁, 31F508α₂, 31F508β₁ and 31F508β₂ are recovered from the fermentation broth at pH 6, mixing with toluene, filtering through a filter press, washing the press with water, loading the aqueous filtrate and washes onto an HP-20 column, eluting the column with 90% methyl alcohol-water and concentrating the methyl alcohol-water giving a crude product.

The crude product is then separated into the α₁, α₂, β₁ and β₂ components and further purified by high performance liquid chromatography on a reverse-phase column equilibrated with 0.1% trifluoroacetic acid in water followed by elution with an acetonitrile gradient to 10%. Active fractions are pooled and freeze dried. Freeze dried fractions are dissolved in 0.01M hydrochloric acid, applied to a reverse phase column and eluted with an acetonitrile gradient to 10% resolves the two components α₁ and α₂. In neutral or basic solution, α₁ and α₂ convert to the beta form. Beta is the quinone form of α₁ or α₂ having lost hydrogens from the oxygens connected to carbon-10 and 13.

The invention will be further described in conjunction with the following non-limiting examples.

EXAMPLE 1 Inoculum Preparation

A typical medium used to grow the primary inoculum is prepared according to the following formula:

    ______________________________________                                         Glucose          1.0%                                                          Dextrin          2.0%                                                          Yeast extract    0.5%                                                          NZ Amine A ®.sup.1                                                                          0.5%                                                          Mississippi line 0.1%                                                          FD-82 defoamer   0.3%                                                          Water qs         100.0%                                                        ______________________________________                                    

This medium is sterilized and 100 ml, in a 500 ml flask, is inoculated with Streptomyces viridodiastaticus subsp. "littus". The medium is then placed on a rotary shaker and incubated at 28° C. for 48 hours providing a primary inoculum. This primary inoculum is then used to inoculate 10 liters of the same sterile medium in a bottle. This medium is grown at 28° C. for 72 hours with a sterile air flow of 10 liters per liter of mash per minute and agitation by an impeller driven at 500 rpm, providing a tertiary inoculum.

EXAMPLE 2 Fermentation

A fermentation medium of the following formulation is prepared:

    ______________________________________                                         Glucose          1.0%                                                          Dextrin          2.0%                                                          Yeast extract    0.5%                                                          NZ Amine A ®.sup.1                                                                          0.5%                                                          Mississippi line 0.1%                                                          FD-82 defoamer   0.3%                                                          Water qs         100.0%                                                        ______________________________________                                    

This medium is sterilized and 1500 liters is then inoculated with the tertiary inoculum of Example 1. The fermentation is conducted at 28° C. with a sterile air flow of 200 liters per minute and agitation by an impeller driven at 200 rpm for 50 hours, at which time the mash is harvested.

EXAMPLE 3 Isolation and Purification of 31F508α₁, 31F508α₂ and 31F508β

The mash is mixed with 7.1 liters of toluene for 30 minutes and filtered through a 36 inch filter press. The press cake is washed with water. The aqueous filtrate and washes are loaded onto a 60L HP20 column. After water washing with two column volumes, the column is eluted with three column volumes of 90% methyl alcohol-water. Twenty liter fractions are taken and assayed. Active fractions are pooled, concentrated and freeze dried. The HP20 column is washed with two column volumes of methyl alcohol. The wash is concentrated to about 3 liters.

The freeze dried material is suspended in methanol and the insolubles removed by filtration. The soluble material is concentrated to dryness and the concentrate dissolved in 1% trifluoroacetic acid in water. Insolubles are removed by centrifugation. The soluble material is loaded onto a preparative reverse phase C18 column equilibrated with 0.1% trifluoroacetic acid in water. The column is eluted with an acetonitrile gradient to 10%. Active fractions containing the alpha component are pooled and freeze dried. Active fractions containing the beta component are adjusted to pH 8 with solid ammonium bicarbonate and extracted with methylene chloride. The beta component is stored in methylene chloride as drying leads to rapid decomposition.

The freeze dried material is dissolved in 0.01M hydrochloric acid and applied to a PRP-1 reversed phase column. The column is eluted with an acetonitrile gradient to 10% affording two components α₁ and α₂. The fractions are freeze dried to off white powders. The total of α₁ isolated is 20 mg. The total of α₂ isolated is 350 mg. In neutral or basic solution, α₁ and α₂ converts to the beta form. Beta is the quinone form of α₁ or α₂ having lost hydrogens from the oxygens connected to carbon-10 and 13. 

What is claimed is:
 1. The compound 31F508α₁ which has the structure: ##STR6##
 2. The compound 31F508α₂ which has the structure: ##STR7##
 3. The compound 31F508β₁ which has the structure: ##STR8##
 4. The compound 31F508β₂ which has the structure: ##STR9##
 5. A method of treating bacterial infections in warm-blooded animals which comprises administering to said animals an antibacterially effective amount of antibiotic 31F508α₁ as defined in claim
 1. 6. A method of treating bacterial infections in warm-blooded animals which comprises administering to said animals an antibacterially effective amount of antibiotic 31F508α₂ as defined in claim
 2. 7. A method of treating bacterial infections in warm-blooded animals which comprises administering to said animals an antibacterially effective amount of antibiotic 31F508β₁ as defined in claim
 3. 8. A method of treating bacterial infections in warm-blooded animals which comprises administering to said animals an antibacterially effective amount of antibiotic 31F508β₂ as defined in claim
 4. 